Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine. (1/194)

The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH7 and pH8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.  (+info)

Transport and utilization of hexoses and pentoses in the halotolerant yeast Debaryomyces hansenii. (2/194)

Debaryomyces hansenii is a yeast species that is known for its halotolerance. This organism has seldom been mentioned as a pentose consumer. In the present work, a strain of this species was investigated with respect to the utilization of pentoses and hexoses in mixtures and as single carbon sources. Growth parameters were calculated for batch aerobic cultures containing pentoses, hexoses, and mixtures of both types of sugars. Growth on pentoses was slower than growth on hexoses, but the values obtained for biomass yields were very similar with the two types of sugars. Furthermore, when mixtures of two sugars were used, a preference for one carbon source did not inhibit consumption of the other. Glucose and xylose were transported by cells grown on glucose via a specific low-affinity facilitated diffusion system. Cells derepressed by growth on xylose had two distinct high-affinity transport systems for glucose and xylose. The sensitivity of labeled glucose and xylose transport to dissipation of the transmembrane proton gradient by the protonophore carbonyl cyanide m-chlorophenylhydrazone allowed us to consider these transport systems as proton symports, although the cells displayed sugar-associated proton uptake exclusively in the presence of NaCl or KCl. When the V(max) values of transport systems for glucose and xylose were compared with glucose- and xylose-specific consumption rates during growth on either sugar, it appeared that transport did not limit the growth rate.  (+info)

Oxythiamine and dehydroepiandrosterone induce a G1 phase cycle arrest in Ehrlich's tumor cells through inhibition of the pentose cycle. (3/194)

Transketolase (TK) reactions play a crucial role in tumor cell nucleic acid ribose synthesis utilizing glucose carbons, yet, current cancer treatments do not target this central pathway. Experimentally, a dramatic decrease in tumor cell proliferation after the administration of the TK inhibitor oxythiamine (OT) was observed in several in vitro and in vivo tumor models. Here, we demonstrate that pentose cycle (PC) inhibitors, OT and dehydroepiandrosterone (DHEA), efficiently regulate the cell cycle and tumor proliferation processes. Increasing doses of OT or DHEA were administered by daily intraperitoneal injections to Ehrlich's ascites tumor hosting mice for 4 days. The tumor cell number and their cycle phase distribution profile were determined by DNA flow histograms. Tumors showed a dose dependent increase in their G0-G1 cell populations after both OT and DHEA treatment and a simultaneous decrease in cells advancing to the S and G2-M cell cycle phases. This effect of PC inhibitors was significant, OT was more effective than DHEA, both drugs acted synergistically in combination and no signs of direct cell or host toxicity were observed. Direct inhibition of PC reactions causes a G1 cell cycle arrest similar to that of 2-deoxyglucose treatment. However, no interference with cell energy production and cell toxicity is observed. PC inhibitors, specifically ones targeting TK, introduce a new target site for the development of future cancer therapies to inhibit glucose utilizing pathways selectively for nucleic acid production.  (+info)

Preparation and chemical composition of the cell walls of Streptococcus mutans. (4/194)

Purified cell walls from Streptococcus mutans strain BHT were prepared without the use of proteolytic enzymes in order to retain all cell wall constituents for chemical analysis. Of four methods employed, the Ribi cell fractionator produced disrupted cell suspensions which could be most thoroughly purified on sucrose gradients. Results of chemical analyses on purified cell walls prepared in this 8.9% glycerol teichoic acid, 33.6% non-peptidoglycan polysaccharide, and 49.9% peptidoglycan.  (+info)

Membrane-bound sugar alcohol dehydrogenase in acetic acid bacteria catalyzes L-ribulose formation and NAD-dependent ribitol dehydrogenase is independent of the oxidative fermentation. (5/194)

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.  (+info)

Epididymal carbohydrate metabolism. III. Metabolism of the caput and cauda epididymidis after separation from the testis in the rat. (6/194)

After unilateral separation of the rat epididymis from the testis, the metabolism of various substrates in vitro by tissue from the attached and separated caput and cauda epididymidis at 7 and 28 days after surgery was determined by radiorespirometry. Hourly collections of 14-CO2 were made during 5-hr incubations. The patterns of 14-CO2 evolution from glucose indicated that most of the metabolic activity followed the Embden-Meyerhof glycolytic and the Krebs cycle respiration pathways. The alteration of the rate of glycolysis was always greater than that of respiration. In all samples, the metabolism of (2-14C) glucose was approximately equal to that of (6-14C) glucose (G-6)and less than that of (1-14C) glucose (G-1). Pentose cycle activity was indicated in all tissues from the caput and cauda epididymidis by the preferential utilization of G-1 over G-6. At 7 and 28 days after surgery, respectively, the G-1:G-6 ratios of 14-CO2 evolution after incubation for 2 hr were 9.75 and 7.79 for the separated caput, 5.17 and 2.66 for the intact caput, 3.11 and 2.52 for the separated cauda and 3.73 and 2.84 for the attached cauda epididymidis. Although epididymal separation did not effect the metabolism of (U-14C) glucose or (U-14C) fructose, glucose appeared to be a more important epididymal substrate than fructose.  (+info)

Formation pathways for lysine-arginine cross-links derived from hexoses and pentoses by Maillard processes: unraveling the structure of a pentosidine precursor. (7/194)

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates and their formation pathways are largely unknown. Synthesis and unequivocal structural characterization are reported for the lysine-arginine cross-links N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S,3R)-2,3,4- trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOGDIC 12), N(6)-(2-([(4S)-4-ammonio-5-oxido-5-oxopentyl]amino)-5-[(2S)-2,3-dihydroxypropyl]- 3,5-dihydro-4H-imidazol-4-ylidene)-l-lysinate (DOPDIC 13), and 6-((6S)-2-([(4S)-4-ammonio-5-oxido-5-oxopentyl] amino)-6-hydroxy-5,6,7,7a-tetrahydro-4H-imidazo[4,5-b] pyridin-4-yl)-l-norleucinate (pentosinane 10). For these compounds, as well as for glucosepane 9 and pentosidine 11, the formation pathways could be established by starting from native carbohydrates, Amadori products, and 3-deoxyosones, respectively. Pentosinane 10 was unequivocally proven to be an important precursor of pentosidine 11, which is a well established fluorescent indicator for advanced glycation processes in vivo. The Amadori products are shown to be the pivots in the formation of the various cross-links 9-13. The bicyclic structures 9-11 are directly derived from aminoketoses, whereas 12 and 13 stem from reaction with the 3-deoxyosones. All products 9-13 were identified and quantified from incubations of bovine serum albumin with the respective 3-deoxyosone or carbohydrate. From these results it seems fully justified to expect both glucosepane 9 and DOGDIC 12 to constitute important in vivo cross-links.  (+info)

Transaldolase deficiency: liver cirrhosis associated with a new inborn error in the pentose phosphate pathway. (8/194)

This article describes the first patient with a deficiency of transaldolase (TALDO1 [E.C.2.2.1.2]). Clinically, the patient presented with liver cirrhosis and hepatosplenomegaly during early infancy. In urine and plasma, elevated concentrations of ribitol, D-arabitol, and erythritol were found. By incubating the patient's lymphoblasts and erythrocytes with ribose-5-phosphate and subsequently analyzing phosphate sugar metabolites, we discovered a deficiency of transaldolase. Sequence analysis of the transaldolase gene from this patient showed a homozygous deletion of 3 bp. This deletion results in absence of serine at position 171 of the transaldolase protein. This amino acid is invariable between species and is located in a conserved region, indicating its importance for enzyme activity. The detection of this new inborn error of pentose metabolism has implications for the diagnostic workup of liver problems of unknown etiology.  (+info)